Viral free semen and methods of producing the same

ABSTRACT

The elimination or reduction of viral contamination of semen used for artificial insemination by incorporating with the semen a diluent or extender containing antibodies for the particular virus or viruses. These extenders containing the antibodies can be serum, milk, egg yolk, culture supernatant or ascites fluid with bovine monoclonal antibodies or gamma globulin fractions of each of the above.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention generally relates to the production of semen forartificial insemination in animals. More specifically, the presentinvention is concerned with a process which will eliminate or reduceinfectious virus contamination in semen and semen free of infectiousvirus thereby produced.

2. Description of the Prior Art

In the artificial insemination of animals, it is well-known that thesemen can potentially be contaminated with different viruses that cancause a variety of diseases. At present, there is no safe or effectiveprocedure to eliminate viral contamination from semen even though it isknown that bacterial contamination of semen can be eliminated or reducedby the addition of antibiotics. No drug presently available willsimilarly eliminate virus without significantly affecting the viabilityof the sperm. (See, e.g. Bartlett et al, "Specific Pathogen Free (SPF)Frozen Bovine Semen: A Goal?", Proc. 6th Tech. Conf. on AI and Repro.N.A.A.B., pp. 11-12 (1976); and Schultz, "When Can We Achieve Our Goalof Providing Specific Pathogen Free Bovine Semen?" U.S.A.H.A. Proc.,81:141-140 (1979)). It has also been suggested that when antibody ispresent as a result of immunization or previous infection in body fluidsor when this antibody, if present in serum, milk or egg yolks, is addedto viruses in vitro rapidly eliminates or reduces their infectivity forsusceptible cells in culture or eliminates infectivity for susceptibleanimals. (See, Bellanti, Immunology II, W. B. Saunders Co.,Philadelphia, PA. (1978). Myrvik and Weisen, Fundamentals of Immunology,Lea & Febiger, Philadelphia, PA (1984)).

Viral contamination of semen has serious implications for the cattleindustry since one infected bull shedding virus in his semen couldpotentially infect cattle regionally, nationally or even internationallythereby leading to quarantine and/or destruction of valuable livestockas a result of infection and/or disease. Artificial insemination and theprocedures used for the performance of artificial insemination serve asa means by which viral contaminated semen could be collected, preserved,stored and disseminated to the cattle population without the knowledgeof the parties involved.

It is apparent that because of the potential danger that viralcontaminated semen presents to the cattle industry, there is a distinctneed for a method which will eliminate or reduce viral contamination ofsemen thus eliminating the problem for both the artificial inseminationindustry and the cattle producer. Although the need has been apparent,this need went unanswered until the present invention.

It has been proposed in the art that semen can be preserved for a periodof time as evidenced by U.S. Pat. Nos. 3,472,735 and 4,329,337. However,as the reader will appreciate from an inspection of those U.S. patents,no solution to the elimination of viral contaminated semen has beensuggested. Thus although such proposals may permit storage of semen, thesemen which is stored may be contaminated with various viruses. (e.g.,Bartlett et al, "Specific Pathogen (SPF) Frozen Bovine Semen: A Goal?",Proc. 6th Tech. Conf. on AI and Repro. N.A.A.B., pp. 11-12 (1976).

BRIEF SUMMARY OF THE INVENTION

According to the present invention, however, a process is provided foreliminating or reducing viral infectivity in contaminated semen.

In accordance with the present invention, it has been found that ifsemen is extended or diluted with serum, milk or egg yolk containingsufficient antibodies specific for contaminating viruses present in thesemen, the viruses would be rendered incapable of infecting cattle whenused in artificial insemination procedures. This extender containing theantibodies, hereinafter referred to by the terminology "immunoextendor",can conceivably be developed for any virus that would contaminate semen.If desired, the "immunoextendor" can contain antibodies specific fornumerous viruses without, once again, affecting the semen viability.

The novel semen product of the present invention is particularly wellsuited for use in artificial insemination. The semen of the presentinvention achieves such beneficial results when it contains an"immunoextendor", the "immunoextendor" containing antibodies which willeffectively eliminate or reduce viral contamination without impeding theviability of fertility of the supernatant or ascites fluid containingbovine monoclonal antibody, or gamma globulin fractions of any or all ofthe above.

Therefore, in accordance with the present invention, a process isprovided which produces semen of the highest quality, free of infectiousviruses and with a high conception rate.

These and other objects and advantages of the present invention willbecome more apparent to the reader after careful consideration is givento the following detailed description of the preferred exemplaryembodiments thereof.

DETAILED DESCRIPTION OF THE PREFERRED EXEMPLARY EMBODIMENTS

It has been found that viral contamination of semen can be eliminated orsubstantially reduced by antibodies without interfering with theviability of the semen. Semen for use in artificial insemination isusually diluted or extended with various diluents to obtain the mosteconomical use thereof. In accordance with the present invention,antibodies specific for various viruses may then be incorporated intothe extender. As used herein and in the accompaning claims, the term"immunoextendor" is meant to refer to a semen extender having viralantibodies incorporated therein.

The addition of the "immunoextendor" to semen intentionally contaminatedwith large amounts of viruses (Infectious Bovine Rhinotracheitis (IBR),Bovine Virus Diarrhea (BVD) and Bluetongue (BT) viruses) eliminatesinfectious virus as determined by inoculating susceptible cell culturesand animals, but does not interfere with sperm viability as determinedby sperm motility and ability of the sperm to cause conception afterartificial insemination. The "immunoextendors" of the present inventioninclude "immunoextendor"-hyperimmune serum, "immunoextendor"-milk,"immunoextendor"-egg yolk, "immunmoextendor"-monoclonal antibody,"immunoextendor"-gamma-globulin. These "immunoextendors" when used inthe process of extending semen for artificial in insemination inaccordance with this invention have been found to reduce or eliminateviral contamination of the semen, rendering it non-infectious when usedto inseminate cattle. Importantly, the "immunoextendor" does notinterfere with the ability of the sperm to cause conception. Theadvantage of using the immunoextendor and the procedure ofimmunoextension is to provide a semen which is free of infectious virusthus providing a more desirable product for artificial insemination.

Hyperimmune serum, hyperimmune milk, hyperimmune egg yolk, gammaglobulin from hyperimmune serum or milk, bovine monoclonal antibodies("Immunoextendor") can be developed for any virus known to contaminatesemen. The "immunoextendor" may, when desirable, contain antibodyactivity to several viruses (e.g., IBR, BVD, BT, foot and mouth disease(FMD) viruses). "Immunoextendors" with multiple reactivity would havebroad spectrum coverage to eliminate any one or more viruses that maycontaminate a specific semen sample. It is proposed that"immunoextendor" be considered similar to antibiotics which arecurrently used by the artificial insemination industry to eliminate orreduce bacterial contamination of semen.

The "immunoextendor" of the present invention is five distinct, butsimilar in purpose, products. (1) The first "immunoextendor" ishyperimmune serum (high antibody titer to the virus or viruses onewishes to eliminate) that is added to the extender (milk or egg yolk)and the semen. The hyperimmune serum can be added to standard milk or toegg yolk extenders to convert them to an "immunoextendor." (2) Thesecond "immunoextendor" is hyperimmune milk, produced by systemic and/orintramammary immunization of non-lacting cows. The immune milk which iscollected shortly after parturition is used as an "immunoextendor" forsemen or can be added to standard milk or to egg yolk extender. (3) Thethird "immunoextendor" is hyperimmune egg yolk. Laying chickens areimmunized with the desired virus and eggs are collected on a daily basisafter high titers of serum antibody are present. The egg yolk iscollected, pooled and an isotonic buffered egg yolk citrate preparationis prepared as the "immunoextendor." (4) The fourth "immunoextendor" isa bovine monoclonal antibody the product of a hybridoma cell producingantibody to the neutralizing antigen of the virus. Mouse monoclonalantibodies are undesirable, because of their inherent antigenicity inthe bovine species. (5) The fifth "immunoextendor" is the gamma globulinfraction of all of the above (described in 1 to 4) prepared by ammoniumsulfate or alcohol precipitation of the same to provide a partiallypurified concentrate of the gamma globulin faction which contains theantibodies.

Studies which have included the "immunoextendors" described above havedemonstrated that semen experimentally contaminated with large amount ofvirus (e.g., IBR, BVD or BT viruses) and extended with "immunoextendor"are free of infectious virus when the semen is placed in tissue cultureor used to inoculate and/or inseminate virus susceptible animals.Furthermore, the "immunoextendors" do not interfere with conception andthe fertility of semen extended with "immunoextendors" is equal to orbetter than non-immunoextendor-treated semen.

The following non-limiting examples will further illustrate and describethe "immunoextendors" of the present invention and their preparation.

EXAMPLE I Hyperimmune Serum

Holstein and Holstein-Angus cross, virgin heifers approximately 1 to 2years old were used to produce hyperimmune serum to IBR, BVD and BTviruses. Commercial intramuscular IBR and BVD vaccine and a laboratoryattenuated BT-serotype 17 virus were used for the antigens. A multipleintramuscular (3 times) followed by intranasal immunization (1 time) wasused to stimulate a high antibody titer. Three months after the lastimmunization, the heifers were treated with 10 mg of dexamethasone(Azium)/day for 5 days, then reimmunized on the fifth day with BVD andBT viruses. Serum was collected on the 7th and 14th day after the BVDand BT viruses were inoculated. The antibody titers were determined bythe serum neutralization test (constant virus as well as constantserum).

The antibody titer for IBR was greater than 1:1000, for BVD greater than1:2,500 and for BTV greater than 1:500. The serum was heat treated at60° C. for 2 hours. This procedure would effectively destroy infectiousvirus, inactivate certain of the complement components and destroyimmunoglobulin E activity, all are unwanted and unneeded components ofthe serum. The serum was tested in aerobic and anaerobic cultureconditions for bacterial contamination. The serum was placed on bovineturbinate cell culture and blind passaged three times to ensure that nocytopathic virus was present. Hyperimmune serum to be used commerciallycould be subjected to ionizing radiation to ensure that virus, ifpresent, would be destroyed.

EXAMPLE II Hyperimmune Milk

Two Holstein cows in the 6th month of gestation were immunizedintramuscularly and intramammary via the teat canal with IBR and BVD.The animals were immunized again at the 8th month in a similar manner.At parturition, the colostrum and the next six milkings were pooled,placed in plastic containers and frozen. A small aliquot of the totalmilk sample was tested for antibody as described above for serum. Theantibody titer to IBR was greater than 1:400 and greater than 1:1000 forBVD virus. The milk was heated to 60° C. for 1 hour and tested forsterility as described above for hyperimmune semen.

EXAMPLE III Hyperimmune Egg Yolk

Eight White Leghorn laying hens were used. Two were hyperimmunized withIBR, two hyperimmunized with BVD and four hyperimmunized with IBR andBVD. Immunization included subcutaneous and intramuscular inoculationduring a one month period. Eggs were collected daily and the yolks wereseparated from the whites, pooled and tested for antibody.

Antibody titer to IBR was approximately 1:100 and for BVD virus wasapproximately 1:500. The egg yolk was prepared as an egg yolk citrateextender.

EXAMPLE IV Monoclonal Antibodies

Bovine x mouse hybridoma would be prepared as previously described(Srikumaran, Guidry and Goldsby. "Bovine X Mouse Hybridomas that SecreteBovine IgG₁ ", Science, Vol. 220, pp. 522-524, 1983, expresslyincorporated hereinto by reference). The monoclonal antibody wasscreened for neutralizing activity to IBR virus. The monoclonal antibodywas added to milk or egg yolk extender to produce "immunoextendor".

EXAMPLE V Gamma Globulin Fraction

Hyperimmune serum, milk, egg yolk and monoclonal antibody was precipatedwith ammonium sulfate or ethanol according to standard procedures toobtain a crude gamma globulin fraction. The gamma globulin fractions aresuperior to all the above preparations because the concentration ofantibodies can be increased, they do not contain unwanted proteinfractions (e.g., albumin), small mounts can be added, and the ethanolwould sterilize the preparation, eliminating possible microbialcontaminants.

Immunoextension

Semen was collected from Holstein and Jersey bulls by standard methods.

Part of the semen from the Holstein bulls and/or the Jersey bulls wascontaminated with various concentrations of IBR and BVD viruses or BTvirus. One-half of the contaminated semen was diluted in extendercontaining hyperimmune serum so that the final diluted semen had 20%serum.

Contaminated semen was also diluted in hyperimmune milk (50%),hyperimmune egg yolk (100%), monoclonal antibody and gamma globulinfractions of hyperimmune serum. The gamma globulin fractions would beequivalent in antibody titer to adding 20%, 50% or 80% whole hyperimmuneserum. The remaining contaminated semen was diluted and frozen in normalextender, (milk or egg yolk citrate with nonimmune serum, or gammaglobulin from a non-immune animal).

Semen Examination

Semen was tested by standard method for numbers and motility.

The ability of the semen extended with hyperimmune serum and gammaglobulin fraction "immunoextendors" to settle cows was tested inexperimental as well as large field trials by standard methods ofartificial insemination.

Results

Results of in vitro and in vivo tests to detect virus in contaminatedsemen are listed in Table 1. Semen contaminated with IBR and BVDextended with hyperimmune serum and hyperimmune milk did not haveinfectious virus which could be detected by either in vitro or in vivotests when concentrations as high as 1×10⁴ TCID₅₀ IBR and BVD virus wereadded to semen. The gamma globulin fraction of hyperimmune serumeliminated virus from the semen containing 1×10⁶ TCID₅₀ of IBR and BVD.The "immunoextendor" was also effective in reducing or eliminatinginfectious bluetongue virus in semen (Table 2).

Sperm motility was good after addition of "immunoextendor". The spermextended with hyperimmune serum when used for insemination was able to"settle" (cause conception) heifers (Table 3). These results clearlydemonstrate that immunoextension with hyperimmune serum or a gammaglobulin fraction thereof and hyperimmune milk are effective in reducingor eliminating virus from semen and that the semen is able to causeconception. The inability of the egg yolk in this trial to similarlyreduce or eliminate virus was caused by the low neutralization titer ofthe egg yolk used but nonetheless the results suggest this method couldalso be used if egg yolk with higher titer antibody to virus was used.Results with monoclonal antibodies suggest they are equal to or betterthan hyperimmune serum.

                                      TABLE 1.                                    __________________________________________________________________________    Results of Viral Infectivity for Semen Contaminated with Infectious           Bovine Rhinotracheitis Virus and                                              Bovine Virus Diarrhea and Extended with Normal Extender or                    "Immunoextendor".                                                             EXTENDER                                                                               Serum               Milk               Egg Yolk                                       Hyper-             Hyper-             Hyper-                                  immune             immune             immune                                  ("Immuno-                                                                            Gamma       ("Immuno-                                                                            Gamma       ("Immuno-              Concentration                                                                          Normal  extendor")                                                                           Globulin                                                                           Normal extendor")                                                                           Globulin                                                                           Normal extendor")             of Virus In  In  In  In In   In  In In  In In   In  In In  In                 (IBR & BVD)                                                                            Vitro.sup.a                                                                       Vivo.sup.b                                                                        Vitro                                                                             Vivo                                                                             Vivo Vitro                                                                             Vivo                                                                             Vitro                                                                             Vivo                                                                             Vivo Vitro                                                                             Vivo                                                                             Vitro                                                                             Vivo               __________________________________________________________________________    1 × 10.sup.3                                                                     .sup. Pos.sup.c                                                                   Pos .sup. Neg.sup.d                                                                   Neg                                                                              Neg  Pos Pos                                                                              Neg Neg                                                                              Neg  Pos Pos                                                                              Neg Pos                1 × 10.sup.4                                                                     Pos Pos Neg Neg                                                                              Neg  Pos Pos                                                                              Neg Neg                                                                              Neg  Pos Pos                                                                              Pos Pos                1 × 10.sup.6                                                                     Pos Pos Neg Pos                                                                              Neg  Pos Pos                                                                              Pos Pos                                                                              Pos  Pos Pos                                                                              Pos Pos                __________________________________________________________________________     .sup.a tissue                                                                 .sup.b animal inoculation                                                     .sup.c virus isolated or seroconversion                                       .sup.d no virus isolated or animals did not seroconvert                  

                  TABLE 2                                                         ______________________________________                                        Results of Viral Infectivity for Semen Contaminated with                      Bluetongue Virus (Serotype 17) Extended with Normal                           Extender or "Immunoextendor".                                                               EXTENDER                                                                      Serum                                                           Concentration                   Gamma                                         of Virus  Normal    Hyperimmune Globulin Fraction                             (TCID50)  In Vivo.sup.a                                                                           In Vivo     In Vivo                                       ______________________________________                                        1 × 10.sup.2                                                                      .sup. Pos.sup.b                                                                         Neg         Neg                                           1 × 10.sup.3                                                                      Pos       Neg         Neg                                           1 × 10.sup.4                                                                      Pos       Neg         Neg                                           1 × 10.sup.5                                                                      Pos       Pos         Neg                                           1 × 10.sup.6                                                                      Pos       Pos         Pos                                           ______________________________________                                         .sup.a Inoculation of susceptible cattle                                      .sup.b Seroconversion Pos. indicates animal became infected. Neg.             indicates no infection                                                   

                  TABLE 3                                                         ______________________________________                                        Effect of Use of "Immunoextendor" (10% Hyperimmune                            Serum and Gamma Globulin) on Conception.                                                               % Pregnant                                           Semen       Treatment    1st Service                                          ______________________________________                                        Hyperimmune Serum (10%)                                                       Bull #1     None         44%                                                  Bull #1     Immunoextendor                                                                             50%                                                  Bull #2     None         56%                                                  Bull #2     Immunoextendor                                                                             56%                                                  Gamma Globulin Fraction (30% Equivalent)                                      Bull #3     None         75%                                                  Bull #3     Immunoextendor                                                                             80%                                                  Bull #4     None         64%                                                  Bull #4     Immunoextendor                                                                             72%                                                  Bull #5     None         76%                                                  Bull #5     Immunoextendor                                                                             75%                                                  Bull #6     None         66%                                                  Bull #6     Immunoextendor                                                                             66%                                                  ______________________________________                                    

As this invention may be embodied in several forms without departingfrom the spirit or essential characteristics thereof, the presentembodiment is, therefore, illustrative and not restrictive, since thescope of the invention is defined by the appended claims rather than bythe description preceding them, and all changes that fall within thescope of the claims or that form their functional as well as conjointlycooperative equivalents are, therefore, intended to be embraced by thoseclaims.

What is claimed is:
 1. A non-infectious semen product for artificialinsemination containing semen and viral antibodies said antibodies beingof a type and being presenting an amount sufficient to reduce oreliminate viruses in the semen product while maintaining the viabilityof the semen.
 2. The semen product of claim 1 wherein the viralantibodies are contained in a serum which is added to a milk or egg yolkcitrate that dilutes the semen.
 3. The semen product of claim 1 whereinthe viral antibodies are contained in milk which dilutes the semen. 4.The semen product of claim 1 wherein the viral antibodies are containedin egg yolk which dilutes the semen.
 5. The semen product of claim 1wherein the viral antibodies are contained in a culture supernatant orascites fluid, as bovine monoclonal antibodies, which is added to themilk or egg yolk citrate that dilutes the semen.
 6. The semen product ofany one of claims 2-5 wherein the viral antibodies are contained in agamma globulin fraction.
 7. The semen product of claim 6 wherein saidgamma globulin fraction is prepared by ammonium sulfate or ethanolprecipication of the serum, milk, egg yolk, culture supernatant orascites fluid.
 8. The semen product of claim 1 wherein the viralantibodies neutralize one or more bovine viruses selected from the groupconsisting of Infectious Bovine Rhinotracheitis, Bovine Diarrhea Virus,Bluetongue Virus and Foot and Mouth Disease Virus.
 9. A method forreducing or eliminating viral contamination in semen used for artificialinsemination comprising incorporating viral antibodies in said semen toform a non-infectious semen product, said antibodies being of a type andbeing present in an amount sufficient to reduce or eliminate viruses insaid semen while maintaining viability of the semen.
 10. The method ofclaim 9 wherein the viral antibodies are contained in a serum whichdilutes the semen.
 11. The method of claim 9 wherein the viralantibodies are contained in milk which dilutes the semen.
 12. The methodof claim 9 wherein the viral antibodies are contained in egg yolk whichdilutes the semen.
 13. The method of claim 9 wherein the viralantibodies are contained in culture supernatant or ascites fluid, asbovine monoclonal antibodies, which dilute the semen.
 14. The method ofany one of claims 10-13 wherein the viral antibodies are contained in agamma globulin fraction.
 15. The method of claim 14 wherein the gammaglobulin fraction is prepared by ammonium sulfate or ethanolprecipitation of the serum, milk, egg yolk, culture supernatant orascites fluid.
 16. The method of claim 9 wherein the viral antibodiesneutralize at least selected one virus from one group of bovine virusesconsisting of Infectious Bovine Rhinotracheitis, Bovine Diarrhea Virus,Bluetongue Virus and Foot and Mouth Disease Virus.
 17. The semen productof claim 1, wherein said semen is bovine semen.
 18. The method of claim9 wherein the semen is bovine semen.